Phorbol esters, which induce cellular differentiation of HL-60 human leukemic cells, decrease the expression of surface transferrin receptors, a membrane component universally associated with rapid cellular proliferation. This alteration of receptors is a rapid event preceding morphological and histochemical differentiation and is associated with increased phosphorylation of the receptor. As such, it may represent an initial signal involved in phorbol ester induced cellular differentiation. The goal of this proposal is to elucidate the mechanism of initial regulation of surface transferrin receptors by tumor promoting phorbol esters. This project will concentrate on biochemical and biological characterization studies directed toward determining the following: (1) evidence that phorbol esters mediate their effect through the enzyme, protein kinase C (PKC). Results from dose-response effects of active phorbol esters will be examined with respect to down-regulation(internalization) of surface transferrin receptors and increased receptor phosphorylation (hyperphosphorylation); (2) that hyperphosphorylation of the receptor directly triggers receptor internalization and may alter subsequent intracellular metabolism of the transferrin receptor; and (3) whether transferrin receptor hyperphosphorylation is directly mediated by PKC. Techniques necessary for determining surface and internalized receptor,and differential receptor phosphorylation will employ radiolabeled ligand binding assays and ligand-affinity chromatography for isolation of the receptor. Characterization of transferrin receptor cycling will be studied following proteolytic removal of surface transferrin binding activity and determining remaining intracellular receptor binding. Bouyant density centrifugation techniques for separating various vesicular compartments will be employed for determining intracellular localization of the receptor. PKC will be purified using ultracentrifugation and anion exchange HPLC techniques. Characterization of in vitro receptor phosphorylation by purified PKC will be performed employing appropriate calcium and phospholipid controls. It is expected that elucidation of the mechanism by which phorbol esters rapidly regulate both the expression of surface transferrin receptors and hyperphosphorylation will provide insight into understanding the role of these key surface receptors in generating initial signals necessary for cellular differentiation